黄癸固体分散体调控AMPK-mTOR信号通路改善糖尿病肾病足细胞损伤的研究

Podocyte injury is crucial for the development and progression of diabetic nephropathy (DN). Podocyte injury during the early DN process is attributed to the autophagy defect and the AMPK-mTOR signal pathway regulates the process of autophagy in podocytes. Therefore, we hypothesize that the dysregulated activation of the AMPK-mTOR pathway is crucial for podocyte injury during the early DN process. Our previous studies have showed that the Huang Kuei-solid dispersion(BSC) nanoparticules we generated can activate the AMPK in the kidney, attenuate podocyte injury, reduce the levels of proteinuria in animals with early DN, and improve renal function. Accordingly, we plan to employ a rat model of high fat-fed and STZ-induced diabetic nephropathy and a cell model of high glucose-induced podocyte injury to examine the levels of the AMPK-mTOR pathway and the autophagy and apoptosis-related protein expression, the formation of autophagy and apoptosis in podocytes using siRNA knockdown, induction of over-expression, and Western blot techniques. Potentially, our data will provide new insights into the role of the AMPK-mTOR pathway in podocyte autophagy and the development of DN. Our findings will reveal the molecular mechanism underlying the therapeutic action of the drug in DN and new therapeutic targets for the development of reagents for the intervention of DN in humans.

足细胞损伤是糖尿病肾病（diabetic nephropathy, DN）发病与进展的中心靶点,DN早期的自噬缺陷是足细胞损伤的重要原因，而AMPK-mTOR信号通路参与细胞自噬形成。为此,推测AMPK-mTOR信号通路异常是引起DN早期足细胞损伤的关键环节。本课题组前期工作表明我们自主研制的黄癸固体分散体纳米载体系统能够激活肾脏AMPK，减轻足细胞损伤，降低DN早期蛋白尿水平，改善肾功能，具有抗DN作用。本课题拟建立高脂饮食联合小剂量STZ腹腔注射诱导的DN大鼠模型及高糖诱导的足细胞损伤模型，利用siRNA干扰及过表达载体等技术，检测足细胞AMPK-mTOR、自噬及凋亡通路关键蛋白表达，观察自噬形成及凋亡情况，旨在阐明AMPK-mTOR信号通路介导的自噬在维持DN足细胞稳定的关键作用，揭示黄癸固体分散体抗DN的分子机制，为DN治疗提供新靶点，也为黄癸固体分体治疗DN提供理论依据。

糖尿病肾病（diabetic nephropathy, DN）为糖尿病最常见、最严重的微血管并发症之一，是引起终末期肾病的首要原因。目前DN尚无特效治疗药物，故探索DN发病机制及寻找安全有效的抗DN新药具有十分重要的意义。足细胞损伤是糖尿病肾病发病与进展的中心靶点,DN早期的自噬缺陷是足细胞损伤的重要原因，而AMPK-mTOR信号通路参与细胞自噬形成。为此，我们认为AMPK-mTOR信号通路异常是引起DN早期足细胞损伤的关键环节。本课题组前期工作表明自主研制的黄癸固体分散体纳米载体系统（黄连素与肠道促吸收剂癸酸钠）能够激活糖尿病大鼠肾脏AMPK，减轻足细胞损伤，降低DN早期蛋白尿水平，改善肾功能而具有抗DN作用。. 基于此，本项目建立了高脂饮食联合小剂量STZ腹腔注射诱导的DN大鼠模型及高糖诱导的足细胞损伤模型并予以黄癸固体分散体（黄连素）干预，利用siRNA干扰、透射电子显微镜、免疫荧光技术、Western blot方法、流式细胞仪技术等，检测足细胞标志蛋白、AMPK-mTOR信号通路、自噬及凋亡通路关键蛋白表达，观察足细胞损伤、自噬形成、凋亡情况。研究结果表明，DN早期存在足细胞损伤、自噬水平增高，激活AMPK-mTOR信号通路可调节足细胞自噬，从而减轻高糖诱导的足细胞损伤；黄癸固体分散体的抗DN有效成分黄连素可通过调控AMPK-mTOR信号通路，增强足细胞自噬，减轻高糖诱导的足细胞损伤，改善肾功能。. 由此，阐明了AMPK-mTOR信号通路介导的自噬在维持DN足细胞稳定的关键作用，揭示了黄癸固体分散体抗DN的分子机制，为DN治疗提供新靶点即增强AMPK活性调节自噬的药物将可能改善糖尿病肾病的足细胞损伤，为黄癸固体分体（黄连素）治疗DN提供了理论依据，也对减轻糖尿病肾病患者病情、降低医疗资源消耗具有潜在的社会价值与经济价值。